Skin loss due to trauma or disease is usually treated by the autograft technique, that is, by substituting the missing skin with pieces taken from donor areas of the same patient. An important step forward in the treatment of such lesions by reconstructive surgery is represented by in vitro cultures of keratinocytes (kc) (J. Rheinwald and H. Green, Cell, 6:331, 1975), whereby said cultures are allowed to expand in vitro, and membranes of epidermal cells are obtained which are potentially useful to cover skin wounds. This technique has been widely used in clinical practice, mainly for burn patients (G. G. Gallico et al., M. Engl.J. Med., 311-448, 1984), but problems arose right from the start, such as the difficulty for such grafts to take, the fragility of the epithelial sheets and consequent difficulty for the surgeon to handle them.
A different approach was adopted by Yannas et al. (Science, 215:174, 1982), who used resorbable porous materials constituted by coprecipitates of collagen and glycosaminoglycans (GAG), particularly chondroitin-6-sulphate, covered by a thin film of silicon membrane. The characteristic of such materials is that they present randomly formed, intercommunicating pores, rather like a sponge.
S. Boyce and J. Hansbrough (Surgery, 103-421, 1988) described growing kc on the surface of membranes made of collagen and GAG, reducing the surface porosity of the material. In order to limit the development of epidermal culture on the membrane surface, an additional, non-porous layer was inserted.
Skin graft technology must take into consideration the interaction between kc, the basal membrane and the underlying dermis. Nowadays it is generally thought that in the case of full-thickness lesions, autografts can be notably facilitated by placing a dermal bed in the wound underneath the epithelial layer.
The basal kc thus lie on a more physiological substrate and can develop a basal membrane and dermal-epidermal linking structures, capable of lending the necessary resistance to the graft.
Recent clinical studies have suggested that Cuono's method (Langdon et al. J. Invest. Dermatol. 91 5: 478, 1988), whereby a "full-thickness" wound is treated with an allograft from the skin of a cadaver, gives better results in terms of the percentage of graft which takes and general quality of the skin on healing.
However, grafts are difficult to obtain, expensive to store and are potential carriers of pathogenic viruses.
There is clearly a need for new, biodegradable, artificial skin substitutes which do not have these drawbacks, and satisfy the following requirements:
Indeed, the products already on the market or being developed present certain drawbacks: their degradation is uncontrollable and interferes with the wound healing process, thus favouring inflammation. Moreover, these substitutes require the epithelial cells to be thickly seeded on the support and left to proliferate for a long time.
Some examples of the products known to date and generally recommended for use in treatment of severe burns are:
However, these, and other products do not allow the in vitro reconstruction of a perfectly functional dermoepidermal junction.
The use of hyaluronic acid (HA) derivatives has also been described (EPA. 0216453) for the preparation of suitable products, particularly membranes to support human kc growth (EPA 0462426) and non-woven tissues (WO 93/11803). Abatangelo et al. (Wound Repair and Regeneration, January-March, p. 80, 1995) reported the use of nonwoven tissue to support mixed cultures of human kc/fibroblasts. Even so, the epithelial layer was not homogeneous because of the kc which seeded in the gaps in the non-woven tissue, thus giving it an uneven thickness. Degradation of the polymers considered here mainly produces hyaluronic acid (HA), which is a normal constituent of the extracellular matrix and therefore has the advantage of being metabolized by normal cell mechanisms.
The chemical modifications allow the product to remain at the site of the graft for far longer than is possible with the natural polymer, and the receptor interactions are maintained throughout that time. In this regard, it must be remembered that the main receptor for HA, known as CD44, is normally expressed in the epithelial tissues, particularly in the basal and spinous layers of the epidermis, while expression of the protein gradually decreases in the upper layers until it disappears in the completely differentiated kc (Carter et al., J. Cell Biol. 113207, 1991). Moreover, the role of CD44 in HA's degradation mechanism is well documented (Culty et al. J. Cell. Biol. 111:2765, 1990; Underhill, Dev. Biol. 155:324, 1993).
The purpose of the present invention is an artificial human skin simulating both the epidermal and dermal layer of the natural one, wherein both fibroblasts and keratinocytes (kc) are present, both cell types actively proliferating and separated, at the interface, by a protein extracellular matrix, having the characteristic of a dermoepidermal junction.